human recombinant dpp4  (R&D Systems)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Dipeptidyl Peptidase 4, a Novel Adipokine, Impairs Retinal Microcirculation in Patients With Type 2 Diabetes Mellitus

doi: 10.1167/iovs.66.14.4

Figure Lengend Snippet: Effect of DPP4 intraluminal incubation on the BK-induced vasodilation of the retinal arterioles ( A ). The dose-dependent effect of DPP4 in response to BK is examined before (control, n = 16), and after intraluminal incubation with 100 ng/mL ( n = 4), 400 ng/mL ( n = 4), or 1 µg/mL ( n = 8) DPP4 for 3 hours ( B ). The time-course effect of DPP4 in response to BK is examined before (control), and after intraluminal incubation with 1 µg/mL DPP4 for 1, 2, and 3 hours ( n = 8). * P < 0.05 versus control.

Article Snippet: Human recombinant DPP4 was purchased from R&D Systems, and DPP4 inhibitor teneligliptin was obtained from Mitsubishi Tanabe Pharma Co., Ltd. (Osaka, Japan).

Techniques: Incubation, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Dipeptidyl Peptidase 4, a Novel Adipokine, Impairs Retinal Microcirculation in Patients With Type 2 Diabetes Mellitus

doi: 10.1167/iovs.66.14.4

Figure Lengend Snippet: Effect of coadministration of DPP4 with superoxide scavenger, NADPH oxidase inhibitor, xanthine oxidase inhibitor, or DPP4 inhibitor ( A ). The dilation of the retinal arterioles to BK is examined before (control, n = 14) and after intraluminal incubation with 1 µg/mL DPP4 plus the superoxide anion scavenger TEMPOL (1 mM; n = 5), the NADPH oxidase inhibitor apocynin (100 µM; n = 4), or the xanthine oxidase inhibitor allopurinol (10 µM; n = 5) ( B ). Dilation of the retinal arterioles in response to BK is examined before (control, n = 5) and after intraluminal incubation with 1 µM plus the DPP4 inhibitor teneligliptin (0.5 µM; n = 5). * P < 0.05 versus control. † P < 0.05 versus DPP4 alone.

Article Snippet: Human recombinant DPP4 was purchased from R&D Systems, and DPP4 inhibitor teneligliptin was obtained from Mitsubishi Tanabe Pharma Co., Ltd. (Osaka, Japan).

Techniques: Control, Incubation

Journal: Small Science

Article Title: Sustained‐Release Sitagliptin Microneedles for Scar Prevention via Fibroblast‐to‐Adipocyte Conversion

doi: 10.1002/smsc.202500140

Figure Lengend Snippet: DPP4 − KMSCs exhibit higher adipogenic potential in vitro. A–D) Keloid tissues were collected, minced, and seeded to isolate and culture keloid‐derived fibroblasts (A). The isolated fibroblasts were then differentiated toward adipogenic (B), osteogenic (C), and chondrogenic fates (D). E) Surface marker expressions were analyzed using flow cytometry. F) Keloid‐derived DPP4 + and DPP4 − fibroblasts were sorted via fluorescence‐activated cell sorting. G) RT‐qPCR was performed to evaluate the mRNA level of OCT4 , MYC , SOX2 , and NANOG . n = 3; *, p < 0.05. H–J) Sorted DPP4 +/− fibroblasts underwent adipogenic induction, followed by Oil Red O staining and quantification analysis (H–I). Ten random fields per sample were analyzed. The mRNA levels of adipogenesis‐related genes were detected using RT‐qPCR (J); n = 3. Scale bars, 100 μm in (A, B, C, D, and H). Data are presented as mean ± SEM; statistical significance was determined by Student's t ‐test.

Article Snippet: To identify the direct truncation of IGF1 by DPP4, recombinant human IGF1 (rhIGF1, 291‐G1, R&D Systems, Minneapolis, MN) was incubated with a recombinant human DPP4 Fc chimera (11141‐SE, R&D Systems). rhDPP4/Fc was diluted to a concentration of 0.2 ng μL −1 in the assay buffer, and 1 μL of the diluted rhDPP4/Fc was added to 15 μL of diluted rhIGF1 (1 μg μL −1 ).

Techniques: In Vitro, Derivative Assay, Isolation, Marker, Flow Cytometry, Fluorescence, FACS, Quantitative RT-PCR, Staining

Journal: Small Science

Article Title: Sustained‐Release Sitagliptin Microneedles for Scar Prevention via Fibroblast‐to‐Adipocyte Conversion

doi: 10.1002/smsc.202500140

Figure Lengend Snippet: DPP4 − KMSCs demonstrate enhanced adipogenic capacity, whereas DPP4 + KMSCs demonstrate increased fibrotic capacity in vivo. FACS‐sorted DPP4 + and DPP4 − cells were subcutaneously transplanted into Balb/c nude mice. A) The volume of subcutaneously transplanted KMSCs in mice was measured, n = 9. B–D) Adipocyte formation was evaluated by (B) H&E staining (black arrow) and (C) immunofluorescence staining of Perilipin and HLA‐ABC. The black arrow indicates the newly formed adipocytes. (D) The numbers of adipocytes and immature adipocytes were analyzed; n = 6. E,F) Masson's trichrome staining and G,H) immunofluorescence staining of COL1A1 were performed to evaluate collagen deposition. The expression of I,J) PPARγ and K,L) CEBPα was examined by immunofluorescence staining. n = 6. **, p < 0.01. Scale bars, 200 μm in (B), 100 μm in (C), 20 μm in (E, G, I, and K). Data were presented as mean ± SEM. Statistical significance was determined by Student's t ‐test.

Article Snippet: To identify the direct truncation of IGF1 by DPP4, recombinant human IGF1 (rhIGF1, 291‐G1, R&D Systems, Minneapolis, MN) was incubated with a recombinant human DPP4 Fc chimera (11141‐SE, R&D Systems). rhDPP4/Fc was diluted to a concentration of 0.2 ng μL −1 in the assay buffer, and 1 μL of the diluted rhDPP4/Fc was added to 15 μL of diluted rhIGF1 (1 μg μL −1 ).

Techniques: In Vivo, Staining, Immunofluorescence, Expressing

Journal: Small Science

Article Title: Sustained‐Release Sitagliptin Microneedles for Scar Prevention via Fibroblast‐to‐Adipocyte Conversion

doi: 10.1002/smsc.202500140

Figure Lengend Snippet: Sitagliptin‐mediated adipogenic promotion is dependent on protection of IGF1 from DPP4 cleavage. A) Schematic of DPP4‐driven peptide/protein truncation. B–D) DPP4 directly truncates IGF1 but not BMP4. Recombinant human IGF‐I or BMP4 was incubated with recombinant human DPP4 Fc chimera for the cleaving assay, and the product was analyzed using HPLC‐MS/MS. (B) The peptide starting with “ETLC” (red arrow) represented the truncated product of IGF1 by DPP4 and (C) peptide starting with “KHHS” represented the truncated product of BMP4 by DPP4. (D) Percentages of truncated product from the cleaving assay were calculated. E–I) KMSCs underwent adipogenic induction and treated with sitagliptin (20 μM) or combined with the IGF‐1 signaling pathway inhibitor picropodophyllin (PPP, 50 nM), the mRNA and protein levels of adipogenesis‐related molecules were detected by RT‐qPCR E) and F–I) western blotting and quantification analysis. n = 3; *, p < 0.05, **, p < 0.01. Cont. medium, control medium, diff. medium, differentiation medium. Sitag, sitagliptin. M, marker. Data was presented as mean ± SEM. Statistical significance was determined by one‐way analysis of variance (ANOVA) followed by Tukey's HSD post hoc test.

Article Snippet: To identify the direct truncation of IGF1 by DPP4, recombinant human IGF1 (rhIGF1, 291‐G1, R&D Systems, Minneapolis, MN) was incubated with a recombinant human DPP4 Fc chimera (11141‐SE, R&D Systems). rhDPP4/Fc was diluted to a concentration of 0.2 ng μL −1 in the assay buffer, and 1 μL of the diluted rhDPP4/Fc was added to 15 μL of diluted rhIGF1 (1 μg μL −1 ).

Techniques: Recombinant, Incubation, Tandem Mass Spectroscopy, Quantitative RT-PCR, Western Blot, Control, Marker

Journal: Small Science

Article Title: Sustained‐Release Sitagliptin Microneedles for Scar Prevention via Fibroblast‐to‐Adipocyte Conversion

doi: 10.1002/smsc.202500140

Figure Lengend Snippet: Illustration of our work. In scar tissues, IGF1 is cleaved by DPP4 enzyme in DPP4 + KMSC. Cleaved IGF1 may induce KMSC differentiation toward myofibroblasts, whereas IGF1 protected by sitagliptin acts as an adipogenic agent.

Article Snippet: To identify the direct truncation of IGF1 by DPP4, recombinant human IGF1 (rhIGF1, 291‐G1, R&D Systems, Minneapolis, MN) was incubated with a recombinant human DPP4 Fc chimera (11141‐SE, R&D Systems). rhDPP4/Fc was diluted to a concentration of 0.2 ng μL −1 in the assay buffer, and 1 μL of the diluted rhDPP4/Fc was added to 15 μL of diluted rhIGF1 (1 μg μL −1 ).

Techniques:

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Overview of the pooled CRISPR screen. The genome-scale Brunello library was introduced into Cas9 expressing Caco2 cells, followed by selection of transduced cells. After 10 days, pooled Caco2 cells were infected with HAstV1 for 24h, followed by staining using anti-HAstV capsid antibody. Uninfected cells were sorted to determine the sgRNA counts by next-generation sequencing. ( B ) FcRn protein levels in Cas9-Caco2 cells disrupted for FCGRT or DPP4 using independent sgRNAs per gene or targeted with a control anti-GFP sgRNA. Two independent replicates are shown. ( C ) Representative histogram for surface expression of DPP4 in naïve Caco2 cells stained with anti-DPP4 antibody or isotype control antibody. ( D ) Representative histogram showing DPP4 expression in fixed, permeabilized Caco2 cells disrupted for FCGRT or DPP4 or B2M using gene specific sgRNAs or targeted with a control anti-GFP sgRNA. ( E ) Percentage of DPP4 expressing Caco2 cells disrupted for DPP4 using sgRNAs or targeted with a control anti-GFP sgRNA. (n=3) ( F ) Percentage of anti-HAstV capsid antibody stained Caco2 cells treated with PBS (n=5) or isotype antibody (n=5) or various concentrations of anti-DPP4 antibody (n=6) prior to HAstV1 infection. ( G ) Percentage of anti-HAstV capsid antibody-stained control (n=6), FCGRT (n=6) or DPP4 (n=6) knockout Caco2 cells transfected with HAstV1 RNA at 48h post-transfection. Results were analyzed using Kruskal-Wallis test with Dunn’s post-test (F and G) from two to three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: CRISPR, Expressing, Selection, Infection, Staining, Next-Generation Sequencing, Control, Knock-Out, Transfection

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Enrichment [-log10 Robust Rank Aggregation (RRA)] scores of positively-selected sgRNAs in HAstV1-negative cells sorted 24hpi of a genome-wide CRISPR-Cas9 Caco2 cell library compared to HAstV1-positive cells, calculated by MAGeCK. ( B,C ) Cas9-Caco2 cells disrupted for FCGRT (n=6-9) or B2M (n=9) using two independent sgRNAs per gene or targeted with a control anti-GFP sgRNA (n=9) were infected with HAstV1or HAstV8, then stained with anti-HAstV capsid antibody at 24hpi. ( D ) Mean fluorescent intensity (MFI) of DPP4 in negative and positive cell populations of Caco2 cells infected with HAstV1 (n=6) or HAstV8 (n=6) infected at 24hpi. ( E ) Cas9-Caco2 cells were disrupted for DPP4 (n=9) using two independent sgRNAs per gene or targeted with a control anti-GFP sgRNA (n=9), then assessed for infection using anti-HAstV capsid antibody 24hpi with HAstV1 or HAstV8. (F ) Percentage of anti-HAstV capsid antibody-stained Caco2 cells treated with PBS (n=4-5) or isotype control (n=4-6) or anti-DPP4 polyclonal antibody (n=6-7) for 12h hours prior to infection with HAstV1 or HAstV8. Results from three independent experiments were analyzed using the Kruskal-Wallis test with Dunn’s post-test (B to F). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: Genome Wide, CRISPR, Control, Infection, Staining

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A,B ) Enrichment scores of positively-selected sgRNAs in HAstV1-or HAstV8-positive cells sorted 24hpi from a surfaceome CRISPRa Caco2 cell library compared to unsorted cells, calculated by MAGeCK. ( C ) dCas9-Caco2 cells were targeted for DPP4 (n=8) using two independent sgRNAs per gene or a control anti-GFP sgRNA (n=8), then assessed for infection using anti-HAstV capsid antibody 24hpi with HAstV1 or HAstV8. ( D, E ) HAstV1 levels in HEK293T (293T) (n=6) or HEK293T stably expressing FCGRT (293T-FCGRT) (n=8) or DPP4 (293T-DPP4) (n=6) at 24hpi. ( F ) HAstV1 levels in H293T-DPP4 cells treated with isotype control (n=7) or anti-DPP4 antibody (n=7) prior to HAstV infection at 24hpi. Results from three independent experiments were analyzed using Kruskal-Wallis test with Dunn’s post-test (C) or Mann-Whitney test (D to F). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: Control, Infection, Stable Transfection, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Schematic of the CRISPR activation surfaceome screen. The human surfaceome library was introduced into dCas9 expressing Caco2 cells, followed by selection of transduced cells. After 10 days, pooled Caco2 cells were infected with HAstV1 or HAstV8 for 24h, followed by staining using anti-HAstV capsid antibody. We sorted top 3% of the HAstV capsid positive cells to determine the sgRNA counts by next-generation sequencing. ( B ) Representative histogram showing expression of DPP4 in dCas9-Caco2 cells transduced with sgRNAs for DPP4 overexpression. ( C ) FcRn protein levels in dCas9-Caco2 transduced with sgRNA for overexpressing FcRn. Two independent replicates are shown. ( D ) FcRn protein levels in 293T and 293T-FCGRT cells. Two independent replicates are shown. ( E ) Representative histogram showing surface expression of DPP4 in 293T cells stained with anti-DPP4 antibody or isotype control antibody. ( F ) Abundance of DPP4 and HAstV capsid positive 293T and 293T-DPP4 cells infected with HAstV1. ( G, H ) HEK293T or HEK293T-DPP4 cells were transfected with FCGRT (n=5) , DPP4 (n=5) and/or B2M (n=5) plasmids then 48h later were infected and stained with anti-HAstV capsid antibody at 24 hpi. Results were analyzed using Kruskal-Wallis test with Dunn’s post-test (G and H) from three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: CRISPR, Activation Assay, Expressing, Selection, Infection, Staining, Next-Generation Sequencing, Transduction, Over Expression, Control, Transfection

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) HAstV1 was tested against immobilized FcRn and mAb 8E7 via surface plasmon resonance. ( B ) Maturation process of HAstV VP90 structural protein. ( C ) VP25 protein from HAstV1 (left) and HAstV8 (right) was immobilized and tested against 2-fold dilutions of FcRn ranging from 8μM to 62.5nM via biolayer interferometry. ( D, E ) HAstV1 levels at 24hpi in Caco2 cells treated with PBS (Mock) (n=3-6), non-specific control protein (n=6-7), or soluble FcRN (s-FCRN) (n=7) or soluble DPP4 (s-DPP4) (n=6) prior to infection. Results from 2-3 independent experiments were analyzed using Kruskal-Wallis test with Dunn’s post-test (F and G). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: SPR Assay, Control, Infection

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A-C ) Gel filtration and SDS-PAGE profiles of HAstV1 VP25 ( A ) HAstV8 VP25 ( B ) and HAstV1 VP34 ( C ). HAstV1 and HAstV8 VP25 were eluted from a HiLoad 16/600 Superdex 200 column and HAstV1 VP34 was eluted from a Superdex 75 Increase 10/300 column. ( D ) Soluble hDPP4 was expressed via transfection in Expi293 cells and eluted via gel filtration on Superdex 200 Increase 10/300 column (left) and further analyzed using MALS (right). The MALS curve (black) is plotted with the derived molecular weight (red) of 191,3000 Dalton ± 0.848%. ( E-G ) Binding experiments testing full-length spike protein from MERS-CoV, ( E ) VP25 from HAstV1 and HAstV8 ( F , left and right, respectively), and VP34 from HAstV1 ( G ) against DPP4. In all cases, each protein was immobilized via biosensor and tested against 2-fold dilutions of DPP4 from either 1μM to 62.5nM ( E, G ) or 1μM to 15.625nM ( F ).

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: Filtration, SDS Page, Transfection, Derivative Assay, Molecular Weight, Binding Assay

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A,B ) HAstV1 and HAstV8 infection at 24hpi in Caco2 and 293T-FCGRT cells treated with PBS (n=6) or nipocalimab (n=6) prior to infection. ( C, D ) HAstV1 and HAstV8 infection at 24hpi in Caco2 or 293T-DPP4 cells treated with PBS (n=6-8) or sitagliptin (n=6-8) prior to infection. ( E ) HAstV1 levels at 24hpi in HIEs differentiated in monolayers on transwell inserts treated with PBS (n=7), nipocalimab (n=9), anti-DPP4 antibody (n=6) and sitagliptin (n=6) prior to infection. Results from three independent experiments were analyzed using Mann-Whitney test (A to D) or Kruskal-Wallis test with Dunn’s post-test (E). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: Infection, MANN-WHITNEY

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Percentage of anti-HAstV capsid antibody-positive Caco2 cells at 24hpi treated with PBS (n=6) or various concentrations of nipocalimab (n=6) prior to HAstV1 infection. ( B ) Percentage of anti-HAstV capsid antibody-positive Caco2 cells treated with PBS (n=6) or vildagliptin (n=6) or teneligliptin (n=6) prior to HAstV1 or HAstV8 infection. ( C ) Fluorescent 7-Amino-4-Methyl Coumarin (AMC) released by DPP4 enzymatic activity in Caco2 cells treated with PBS (Control; n=4) or DMSO or DPP4 inhibitors (n=4). Results were analyzed using Kruskal-Wallis test with Dunn’s post-test ( A ) from three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: Infection, Activity Assay, Control